The free radical nitric oxide (NO) has been shown in numerous studies, to activate the Ras protooncoprotein. The action of NO on Ras is similar to that of guanine nucleotide exchange factors (GEFs) in that both GEFs and NO modulate Ras activity by promoting guanine nucleotide exchange and populating of Ras in its active Ras-GTP state. Once activated by NO, Ras can transduce signals through a multitude of pathways, leading to changes in cellular growth control. In addition to NO, reactive oxygen species (ROS) (i.e., superoxide anion radical (O2-.) and hydroxyl radical (OH.) have been shown to enhance the rate of Ras guanine nucleotide dissociation in vitro and modulate Ras signaling in vivo. Although NO and ROS are believed to be important regulators of Ras activity, little to known about the mechanism of NO- and ROS-mediated guanine. nucleotide exchange (GNE) on Ras. We have recently postulated a novel mechanism by which NO and O2-. promote Ras activation, which is likely to be common to other redox-active Ras superfamily GTPases. The main goal of this proposal is to investigate the structural and biochemical details of NO-and O2-.-mediated GNE on Ras and Ras-related proteins to both evaluate and further delineate structural and kinetic details associated with the proposed mechanism. To achieve these goals, a multidisciplinary approach employing structural, biochemical and, biophysical methods will be employed. Three main specific aims are proposed: Investigate the structural basis of NO- and O2-.-mediated GNE on Ras and Ras-related GTPases using Nuclear Magnetic Resonance (NMR) spectroscopy. Investigate the structural-basis and kinetic mechanism(s) of NO and O2-.-mediated GNE on Ras and Ras-related GTPases in the presence and absence of GTPase GNE factors using kinetic, biochemical and spectroscopic approaches. Delineate the mechanism of the NO and O2-.-mediated Ras and Ras-related GTPase GNE using Electron Paramagnetic Resonance (EPR) spectroscopy. Understanding the structural, kinetic, and spectroscopic features of the GTP/GDP-binding cycle in the presence and absence of the Ras-activity regulators will aid in understanding how Ras is regulated in the cell and may provide a molecular and mechanistic basis for developing Ras-mediated tumor therapies.